A kind of method that full suspension cell culture produces PRV antigen
Technical field
The invention belongs to veterinary biologicses technical field, and in particular to a kind of full suspension cell culture production pseudorabies
The method of viral antigen.
Background technology
Cell culture invitro production virus is means the most frequently used both at home and abroad, wherein, traditional rolling bottle cell culture process
There is the shortcomings of cell density is low, virus yield is low, production cost is high, labor intensity is big, gradually suspend and train for emerging cell
The technology of supporting is replaced.Cell suspension cultures technology with its automation, scale, cell culture summary, safe operation, batch between
The advantages such as homogenization compensate for numerous deficiencies of traditional spinner culture technology, solve artificial, cost in actual production process, field
The problems such as ground, production cycle, control with raw material, quality be unstable.
Suspension culture techniques are that high density large-scale culture zooblast is used to give birth in bioreactor, under artificial condition
The technology of Tetramune production, it is whether adherent according to cell, it is divided into full suspension cell culture and attached cell microcarrier suspension culture.
Cell microcarrier suspension culture is that cell attachment is suspended in culture in nutrient solution on microcarrier, then by stirring system.Carrier
Substantially or adhere-wall culture, technical difficulty is low, it is adaptable to be difficult to the cell for being domesticated for suspending entirely, and carrier culture exists for culture
There is great birth defect in terms of cost, cell density, ease-to-operate and industry amplification, its maximum volume of culture is no more than
300 liters, which has limited the large-scale application of the technology.Full suspension cell culture need not be by microcarrier, can be continuous close
Carried out in the system closed, reduce the chance of operating procedure and pollution, realized linearisation amplification production, obtaining maximum production
The quality of product can be steadily improved simultaneously, therefore, producing viral vaccine using full suspended cell culture technic has great answer
Use prospect.Such as Publication No.:A kind of A of CN 104027798 Chinese patent application " full suspension cell culture production pig circular ring virus 2
With PK-15B1 cells, suspension free serum culture produces PCV2 antigens to the method for malicious 2 type antigens " entirely, can obtain high yield and quality
Porcine circovirus 2 type antigen.
PRV (Pseudorabies virus, PRV) belongs to herpesviral subfamilies pig blister in herpetoviridae
The type of exanthema virus I, can cause the porcine pseudorabies (Aujeszky ' s disease) of acute infection.Porcine pseudorabies are in world wide
Inside it is very popular, the pig industry to the whole world is very harmful, causes huge economic loss.Pig occurs after pseudoabies, and it is clinical
Symptom is depending on the age of infected pigs, the virulence of Strain, the dosage and approach infected.Adult Pig is general in subclinical infection, bosom
Pregnant sow can cause miscarriage, stillborn foetus, mummy and boar infertility etc. to integrate syndrome.Piglet death rate of the onset within 15 ages in days
Up to 100%, the weanling pig incidence of disease 40%, the death rate 20% or so;Growth retardation can be caused to adult big porker, weightening is slow
Deng.This current disease is considered as to threaten Swine Production and cause one of dead principal disease.
At present, domestic cell in vitro production PRV antigen mainly uses spinner culture method, and minority is using micro-
Carrier suspension culture method.Wherein, rolling bottle technique productions semi-finished product antigen valence is relatively low, and only 105.0-6.8TCID50/ mL, it is necessary to
High power concentration is carried out, and production cycle length is, it is necessary to 10 days.And microcarrier suspension culture technique, though it is improved compared with rolling bottle technique, still
There is larger room for improvement, its production cycle still needs to 6~7 days, and cost is higher, it is difficult to realize and expand culture.At present, without profit
The relevant report of PRV antigen is produced with full suspended cell culture technic.
The content of the invention
To solve technical problem present in prior art, it is an object of the invention to provide a kind of full suspension cell culture
The method for producing PRV antigen.
The technical scheme that the present invention is provided is as follows:
The present invention provides a kind of method that full suspension cell culture produces PRV antigen, and it includes following step
Suddenly:
(1) seed cell BHK21 is recovered, expanded with flask suspension culture, be then seeded to stirring-type biological anti-
Answer in device using the progress suspension culture of low blood serum medium, obtain suspended culture cell liquid;
(2) suspension cell in bioreactor is stopped into stirring and low temperature settles 18~24h, then extract low in supernatant out
Blood serum medium simultaneously inputs serum free medium, continues to cultivate 1~2d, then PRV kind venom is inoculated with reactor, carry out
Virus culture;
(3) continue to cultivate BHK21 96~144h of suspension cell in bioreactor, until dead cell quantity reaches 90%
More than, harvesting suspension, freeze thawing 3 times, 4000rpm centrifugation 5min collect supernatant, produce virus liquid;
(4) virus liquid of harvest is concentrated, inactivated, it is degerming, produce.
Further, above-mentioned steps (1) are recovered seed cell BHK21, are specially with flask suspension culture amplification:
BHK21 cell kinds are taken out into recovery from liquid nitrogen container, added in the DMEM culture mediums containing 10% NBCS, in 37 DEG C, 5%
CO2Lower culture, until it grows up to good individual layer, then with appropriate tryptic digestive juice containing 0.02%EDTA, 37 DEG C digest 6~
8min, is 3~5 × 10 using the DMEM culture mediums adjustment cell density containing 10% NBCS5Individual/mL cell suspension,
It is inoculated in shaking flask, adds the nutrient solution containing 2~4% NBCSs and carry out suspension culture, until cell density reaches 2~4
×106Individual/mL.
Further, the condition of the flask suspension culture is:Shaking flask is placed in 37 DEG C, 5%CO2Trained in incubator
Support, rotating speed is set to 130~150rpm/min;
Further, the nutrient solution for containing 2~4% NBCSs is containing the sub- oleoyl phospholipids of 0.4~1.2mg/L bis-
Choline, 20~40g/L NBCS, the soft phosphatide of 6~12mg/L, the DMEM/F12 (1 of 4~6% (m/v) growth promoters:1) cultivate
Liquid.
Further, the step (1) is seeded in stirring type bioreactor is suspended using low blood serum medium
Culture is specially:Treat cell length in shaking flask to 2~4 × 106Individual/mL, stops shaking flask, and 2000rpm centrifugation 5min are discarded
Clearly, add low blood serum medium and adjust cell density to 3~5 × 105Individual/mL, is then inoculated in stirring type bioreactor,
Under the conditions of low blood serum medium, in 37 DEG C, 5%CO2, pH7.0~7.4, dissolved oxygen DO30~60%, mixing speed 100~
120r/min proceeds the culture that suspends.
Further, the low blood serum medium is to contain 0.2~0.6% (m/v) plant protein peptide, 0.5~1.0%
(m/v) FBS, 0.1~0.5% (m/v) isoflavones, 1.0~1.2% (v/v) are dual anti-, 1~3% (m/v) growth promoter
DMEM/F12 (1:1) nutrient solution.
Preferably, the low blood serum medium also contains 2~4% (m/v) polyglutamic acids.
Further, the step (2) is specially:Cell length in question response device is to 2~4 × 106Individual/mL, will react
Suspension cell stops stirring and 18~24h is settled at 4~6 DEG C in device, then extracts the low blood serum medium and defeated in supernatant out
Enter serum free medium, continue to cultivate 1~2d, be then inoculated with PRV kind venom in the ratio of cell suspension volume 1%, carry out disease
Viral level is 10 in poison culture, described kind of venom5.2TCID50/mL。
Further, the serum free medium is to contain 0.6~1.2% (m/v) plant protein peptide, 0.2~0.6%
(m/v) isoflavones, 1.5~3.5% (m/v) polyglutamic acids, 1.0~1.2% (v/v) are dual anti-, 4~6% (m/v) growths promote
Enter the DMEM/F12 (1 of agent:1) nutrient solution.
Growth promoter of the present invention is by keratan sulfate oligosaccharide and active mineral yeast polypeptides with 1:0.05~
0.2 mass ratio composition, it is preferable that described growth promoter by keratan sulfate oligosaccharide and active mineral yeast polypeptides with
1:0.15 mass ratio composition.
Further, the step (4), which concentrates, is specially:Virus liquid is concentrated by 50K hollow fiber columns;
Inactivation is specially:Using Formalin inactivation, the formalin in viral concentration liquid final concentration of 0.1%;
It is degerming to be specially:Concentrate after inactivation is used into the cylindrical filter cartridge refined filtration that filtering accuracy is 0.45 μm, then it is used
The cylindrical filter cartridge filtration sterilization that precision is 0.20 μm is filtered, sterile working packing is produced.
Further, the Streptomycin Solution of above-mentioned dual anti-penicillin and 10mg/mL for containing 100IU/mL.
Further, it is above-mentioned to take PRV antigen made from full suspension cell culture to add a certain amount of assistant
Finished product vaccine is made in agent emulsification.
The present inventor in experiments it is found that, will using containing 10% NBCS DMEM medium cultures BHK21 it is thin
Born of the same parents' amplification reaches 2~4 × 10 to cell density6Individual/mL, is directly added into low blood serum medium and carries out suspension culture, BHK21 cells
The speed of growth it is slow, cytoactive declines, and low blood serum medium is unable to the growth of support suspension BHK21 cells continually and steadily.
The present inventor is had been surprisingly found that by substantial amounts of experiment, is using the DMEM culture mediums containing 10% NBCS to BHK21 cells
After being cultivated, add the nutrient solution containing 2~4% NBCSs and carry out transition culture, BHK21 cells can be significantly improved low
The growth conditions of blood serum medium, especially when containing a certain amount of growth promoter in described nutrient solution, improved effect is more
It is good, achieve unexpected technique effect.
During the present invention suspends culture production PRV antigen with BHK21 cells entirely, the low blood used
Clear culture medium and serum free medium are added by keratan sulfate oligosaccharide and active mineral yeast polypeptides with certain mass ratio
The growth promoter of composition, described growth promoter can be such that BHK21 cells are kept in the environment of low serum even serum-free
Preferably growth conditions, promote breeding of the virus in cell, substantially increase production efficiency.Described active mineral yeast is more
The complex compound that peptide (ACB Bio-Chelate5) is mineral element with yeast polypeptides formation, the specially zinc of yeast polypeptides complexing,
Copper, magnesium, iron and silicon, are that BHK21 cells and viral growth are numerous purchased from Ai Ti scientific & technical corporation of the U.S. (Active Concepts)
Grow the breeding for promoting cell and virus there is provided essential nutrient.Described keratan sulfate oligosaccharide is with oligomeric
The form of sugar is present, and is conducive to cell to absorb, and it has the similar effect of some growth factors, can stimulate cell proliferation with dividing
Change, improve BHK21 cells and suspend full culture density.
Specifically, described keratan sulfate oligosaccharide is keratan sulfate oligosaccharide mixture, as keratan sulfate
Disaccharides, keratan sulfate trisaccharide, keratan sulfate tetrose, the mixture of keratan sulfate pentasaccharides, its preparation method is with reference to open
Number be CN 1174557A Chinese patent " keratan sulfate oligosaccharide fraction and the medicament containing the fraction ", be specially:Take sulfuric acid
Keratan 50g, is dissolved in 300ml 0.1M acetate buffer solutions (pH6.0), adds-β-N- acerylglucosamine enzymes in 25U
Type sulfuric acid digestive enzyme, degrade 24h at 37 DEG C.After reaction terminates, the ethanol of 2 times of volumes is added, stirring places 1 at room temperature
At night, 15min is centrifuged under 4000rpm, take supernatant (supernatant A).300ml distilled water is added into precipitation, is dissolved, 3 are added
The ethanol of amount, stirring, place centrifuge 15min under 1 night, 4000rpm at room temperature, take supernatant (supernatant B) again.By supernatant A and upper
Clear B mixing, is concentrated under reduced pressure, using Bio-Gel-P-2 posts (3.6 ╳ 134cm), using distilled water as solvent, carries out gel filtration,
Filtrate freeze-drying is produced.Preferably, the present inventor is improved according to method disclosed above, to adjust keratan sulfate
Keratan sulfate disaccharides, keratan sulfate trisaccharide, keratan sulfate tetrose, the ratio of keratan sulfate pentasaccharides in oligosaccharide.Through
Experiment, the inventors discovered that the content of keratan sulfate disaccharides is higher in keratan sulfate oligosaccharide, is more beneficial for BHK21 cells
Grown in low serum even serum free medium, promote virus breeding.
A kind of material that polyglutamic acid is rich in for fermentation soya bean, the present inventor is had found by many experiments, in the training of low serum
Support in base and serum free medium and add a certain amount of polyglutamic acid, the state of BHK21 cell suspension growths can be effectively improved, carried
High cell viability, reduces the appearance of dead cell, promotes cell growth, BHK21 cells is trained in low blood serum medium and serum-free
The cell density for supporting the culture that suspended in base reaches 2~4 × 106Individual/mL, or even this level can be exceeded, to produce the pig of high concentration
Pseudorabies virus antigen vaccine has established technical foundation.
Compared with prior art, advantage of the invention is that:
(1) the PRV antigen that the present invention is produced using full suspension cell culture, is trained with traditional rolling bottle cell
Technique and microcarrier suspension culture technics comparing are supported, production efficiency is substantially increased, and is easy to be enlarged culture, reduction production
Cost, improves product quality, lifts the security of vaccine.
(2) present invention carries out cell suspension cultures using serum free medium, can preferably support the normal of BHK21 cells
Growth, makes its culture that suspends in the reactor comparatively fast to reach higher cell density, and keeps good growth activity, makes a living
The PRV antigen vaccine of production high concentration has established technical foundation, the PRV antigen vaccine potency of gained
Height, it is stable 108.5TCID50/ more than mL, and purity is high, steady quality significantly improves product quality.
Embodiment
The present invention is further described below by way of embodiment, but the present invention is not limited only to following examples.
The keratan sulfate oligosaccharide of embodiment 1 is prepared and constituent analysis
The preparation of A groups:
Keratan sulfate 50g is taken, is dissolved in 300ml 0.1M acetate buffer solutions (pH6.0), 25U mixed enzymes are added,
Degrade 24h at 37 DEG C.Reaction terminate after, add 2 times of volumes ethanol, stirring, placed for 1 night at room temperature, under 4000rpm from
Heart 15min, takes supernatant (supernatant A).300ml distilled water is added into precipitation, is dissolved, the ethanol of 3 times of amounts is added, stirred,
Place at room temperature and centrifuge 15min under 1 night, 4000rpm, take supernatant (supernatant B).Supernatant A and supernatant B is mixed, is concentrated under reduced pressure,
Using Bio-Gel-P-2 posts (3.6 ╳ 134cm), using distilled water as solvent, gel filtration is carried out, is by filtrate freeze-drying
.
The preparation of B-D group keratan sulfate oligosaccharides refers to A groups.
The constituent analysis of keratan sulfate oligosaccharide
Keratan sulfate oligosaccharide made from above-mentioned A-D groups is respectively adopted into ion-exchange chromatography to be separated, taken respectively
Keratan sulfate disaccharides, keratan sulfate trisaccharide, keratan sulfate tetrose, keratan sulfate pentasaccharides fraction are obtained, it is freeze-dried
Afterwards, analyzed using high performance liquid chromatography, detect the content of each composition, as a result see the table below shown:
The content of each oligosaccharide in oligosaccharide made from the 50g keratan sulfates of table 1
Constitute (content %) |
Keratan sulfate disaccharides |
Keratan sulfate trisaccharide |
Keratan sulfate tetrose |
Keratan sulfate pentasaccharides |
A |
20.8 |
12.6 |
6.2 |
2.5 |
B |
23.6 |
13.2 |
4.7 |
2.2 |
C |
22.7 |
12.8 |
5.5 |
2.0 |
D |
20.2 |
12.4 |
5.7 |
2.8 |
Influence of the keratan sulfate oligosaccharide of embodiment 2 to BHK-21 cell growths
The obtained keratan sulfate oligosaccharide of embodiment 1A-D groups is respectively adopted to cultivate BHK21 cells, is specially:
BHK21 cell kinds are taken out into recovery from liquid nitrogen container, added in the DMEM culture mediums containing 10% NBCS,
37 DEG C, 5%CO2Lower culture, until it grows up to good individual layer, then with appropriate tryptic digestive juice containing 0.02%EDTA, 37
DEG C digestion 6min, using containing 10% NBCS DMEM culture mediums adjustment cell density be 5 × 105Individual/mL cell hangs
Liquid, is inoculated in shaking flask, adds the nutrient solution containing 2% NBCS in 37 DEG C, 5%CO2Cultivated in incubator, rotating speed
It is set to carry out suspension culture under the conditions of 150rpm/min, until cell density reaches 4 × 106Individual/mL, wherein, it is newborn containing 2%
The nutrient solution of cow's serum is containing the sub- oleoyl phospholipid choline of 1.0mg/L bis-, the soft phosphatide of 20g/L NBCS, 10mg/L, 6% (m/v)
The DMEM/F12 (1 of growth promoter:1) nutrient solution, described growth promoter is the obtained sulphur of embodiment 1A (B or C or D) groups
Tamarind quality oligosaccharide and active mineral yeast polypeptides are with 1:After 0.15 mass ratio composition, culture 48h, the growth of cell is counted
Density, as a result see the table below 2.
Influence of the keratan sulfate oligosaccharide of the different groups of table 2 to BHK-21 cell growths
Group |
Cultivate cell density (individual/ml) after 48h |
A groups |
1.95×106 |
B groups |
2.20×106 |
C groups |
2.14×106 |
D groups |
1.90×106 |
The content of keratan sulfate disaccharides is higher in upper table 2, keratan sulfate oligosaccharide, is more beneficial for BHK21
Cell grows in the nutrient solution of 2% NBCS, cell is cultivated the density after 48h in rolling bottle and reaches 2.20 × 106
Individual/ml.
The full suspension cell culture of embodiment 3 produces PRV antigen
(1) seed cell BHK21 is recovered, expanded with flask suspension culture:By BHK21 cell kinds from liquid nitrogen container
Recovery is taken out, is added in the DMEM culture mediums containing 10% NBCS, in 37 DEG C, 5%CO2Lower culture, until its grow up to it is good
Good individual layer, then with appropriate tryptic digestive juice containing 0.02%EDTA, 37 DEG C of digestion 6min, using containing 10% NBCS
DMEM culture mediums adjustment cell density be 5 × 105Individual/mL cell suspension, is inoculated in shaking flask, adds and contains 3% new born bovine
The nutrient solution of serum is in 37 DEG C, 5%CO2Cultivated in incubator, rotating speed is set to be suspended under the conditions of 150rpm/min
Culture, until cell density reaches 4 × 106Individual/mL, wherein, the nutrient solution containing 3% NBCS is to contain 1.0mg/L bis-
Sub- oleoyl phospholipid choline, the soft phosphatide of 30g/L NBCS, 10mg/L, the DMEM/F12 (1 of 6% (m/v) growth promoter:1) cultivate
Liquid, described growth promoter is by keratan sulfate oligosaccharide made from embodiment 1B groups and active mineral yeast polypeptides with 1:
0.15 mass ratio composition.
(2) the BHK21 cells of flask suspension culture are seeded in stirring type bioreactor and use low blood serum medium
Carry out suspension culture:Treat cell length in shaking flask to 4 × 106Individual/mL, stops shaking flask, and 2000rpm centrifugation 5min discard supernatant,
Add low blood serum medium and adjust cell density to 5 × 105Individual/mL, is then inoculated in stirring type bioreactor, in low blood
Under clear culture medium condition, in 37 DEG C, 5%CO2, pH7.2, dissolved oxygen DO50%, mixing speed 100r/min proceed suspend
Culture, wherein, low blood serum medium is to contain 0.4% (m/v) plant protein peptide, 1.0% (m/v) FBS, 0.4% (m/v) soybean
Isoflavones, 1.0% (v/v) are dual anti-, 2% (m/v) growth promoter, the DMEM/F12 (1 of 4% (m/v) polyglutamic acid:1) cultivate
Liquid, described growth promoter is by keratan sulfate oligosaccharide made from embodiment 1B groups and active mineral yeast polypeptides with 1:
0.15 mass ratio composition.
(3) the cell length in question response device is to 4 × 106Individual/mL, stirring is stopped and at 4 DEG C by suspension cell in reactor
Lower sedimentation 24h, then extracts the low blood serum medium in supernatant out and inputs serum free medium, continue to cultivate 2d, then by thin
The ratio inoculation PRV kind venom of born of the same parents' suspension volume 1%, carrying out viral level in Virus culture, described kind of venom is
105.2TCID50/ mL, wherein, the composition of serum free medium is to contain 0.8% (m/v) plant protein peptide, 0.4% (m/v) soybean
Isoflavones, 2.0% (m/v) polyglutamic acid, 1.0% (v/v) are dual anti-, 6% (m/v) growth promoter DMEM/F12 (1:1) train
Nutrient solution, described growth promoter is by keratan sulfate oligosaccharide made from embodiment 1B groups and active mineral yeast polypeptides with 1:
0.15 mass ratio composition.
(4) in bioreactor continue cultivate BHK21 suspension cell 120h, until dead cell quantity reach 90% with
On, harvesting suspension, freeze thawing 3 times, 4000rpm centrifugation 5min collect supernatant, produce virus liquid;
(5) virus liquid of harvest is concentrated by 50K hollow fiber columns, then adds formalin and gone out
It is living, wherein, the concentrate after inactivation is finally used filtering by formalin in final concentration of 0.1% (v/v) of viral concentration liquid
Precision is 0.45 μm of cylindrical filter cartridge refined filtration, then with the cylindrical filter cartridge filtration sterilization that filtering accuracy is 0.20 μm, sterile working divides
Dress, is produced.
The full suspension cell culture of embodiment 4 produces PRV antigen
(1) seed cell BHK21 is recovered, expanded with flask suspension culture:By BHK21 cell kinds from liquid nitrogen container
Recovery is taken out, is added in the DMEM culture mediums containing 10% NBCS, in 37 DEG C, 5%CO2Lower culture, until its grow up to it is good
Good individual layer, then with appropriate tryptic digestive juice containing 0.02%EDTA, 37 DEG C of digestion 8min, using containing 10% NBCS
DMEM culture mediums adjustment cell density be 3 × 105Individual/mL cell suspension, is inoculated in shaking flask, adds and contains 2% new born bovine
The nutrient solution of serum is in 37 DEG C, 5%CO2Cultivated in incubator, rotating speed is set to be suspended under the conditions of 140rpm/min
Culture, until cell density reaches 3 × 106Individual/mL, wherein, the nutrient solution containing 2% NBCS is to include 0.6mg/L
Two sub- oleoyl phospholipid choline, the soft phosphatide of 20g/L NBCS, 10mg/L, the DMEM/F12 (1 of 4% (m/v) growth promoter:1) train
Nutrient solution, described growth promoter is by keratan sulfate oligosaccharide made from embodiment 1B groups and active mineral yeast polypeptides with 1:
0.2 mass ratio composition.
(2) the BHK21 cells of flask suspension culture are seeded in stirring type bioreactor and use low blood serum medium
Carry out suspension culture:Treat cell length in shaking flask to 3 × 106Individual/mL, stops shaking flask, and 2000rpm centrifugation 5min discard supernatant,
Add low blood serum medium and adjust cell density to 3 × 105Individual/mL, is then inoculated in stirring type bioreactor, in low blood
Under clear culture medium condition, in 37 DEG C, 5%CO2, pH7.4, dissolved oxygen DO60%, mixing speed 120r/min proceed suspend
Culture, wherein, the composition of low blood serum medium is to include 0.6% (m/v) plant protein peptide, 0.5% (m/v) FBS, 0.2%
(m/v) isoflavones, 1.2% (v/v) be dual anti-, 2% (m/v) growth promoter, the DMEM/F12 of 4% (m/v) polyglutamic acid
(1:1) nutrient solution, described growth promoter is by keratan sulfate oligosaccharide made from embodiment 1B groups and active mineral yeast
Polypeptide is with 1:0.2 mass ratio composition.
(3) the cell length in question response device is to 3 × 106Individual/mL, stirring is stopped and at 6 DEG C by suspension cell in reactor
Lower sedimentation 24h, then extracts the low blood serum medium in supernatant out and inputs serum free medium, continue to cultivate 2d, then by thin
The ratio inoculation PRV kind venom of born of the same parents' suspension volume 1%, carrying out viral level in Virus culture, described kind of venom is
105.2TCID50/ mL, wherein, the composition of serum free medium is big to include 1.2% (m/v) plant protein peptide, 0.5% (m/v)
Beans isoflavones, 3.5% (m/v) polyglutamic acid, 1.0% (v/v) are dual anti-, 4% (m/v) growth promoter DMEM/F12 (1:1)
Nutrient solution, described growth promoter by keratan sulfate oligosaccharide made from embodiment 1B groups and active mineral yeast polypeptides with
1:0.2 mass ratio composition.
(4) in bioreactor continue cultivate BHK21 suspension cell 144h, until dead cell quantity reach 90% with
On, harvesting suspension, freeze thawing 3 times, 4000rpm centrifugation 5min collect supernatant, produce virus liquid;
(5) virus liquid of harvest is concentrated by 50K hollow fiber columns, then adds formalin and gone out
It is living, wherein, the concentrate after inactivation is finally used filtering by formalin in final concentration of 0.1% (v/v) of viral concentration liquid
Precision is 0.45 μm of cylindrical filter cartridge refined filtration, then with the cylindrical filter cartridge filtration sterilization that filtering accuracy is 0.20 μm, sterile working divides
Dress, is produced.
It is prepared by the PRV antigen finished product vaccine of embodiment 5
(1) prepared by oil phase:94 parts of high-quality injection white oil is taken, 2 parts of aluminum stearate, 4 parts of Arlacel-80 is first slow by white oil
Heating, adds Arlacel-80 and aluminum stearate, is heated when stirring, until aluminum stearate be fully dissolved to it is transparent untill, autoclaving
It is standby;
(2) prepared by aqueous phase:4 parts of the Tween-80 that 96 parts of vaccine antigen made from Example 3 is added after sterilizing, starts to stir
Mix, make untill Tween-80 is completely dissolved, aqueous phase is made;
(3) emulsify:Aqueous phase is added in oil phase, emulsified using IKA emulsifying agents, 16000rpm, emulsification 5 minutes is
Can;
(4) dispense:The vaccine of preparation is dispensed according to every bottle of 250mL.
The full suspension cell culture of comparative example 1 produces PRV antigen
The step of full suspension cell culture of comparative example 1 produces PRV antigen is substantially the same manner as Example 3, difference
It is, without using the nutrient solution containing 3% NBCS in the step (1), but uses containing 10% NBCS
DMEM culture mediums carry out Shaking culture to BHK21 cells, are specially:Seed cell BHK21 is recovered, is suspended and trained with shaking flask
Support amplification:BHK21 cell kinds are taken out into recovery from liquid nitrogen container, added in the DMEM culture mediums containing 10% NBCS,
37 DEG C, 5%CO2Lower culture, until it grows up to good individual layer, then with appropriate tryptic digestive juice containing 0.02%EDTA, 37
DEG C digestion 8min, using containing 10% NBCS DMEM culture mediums adjustment cell density be 5 × 105Individual/mL cell hangs
Liquid, is inoculated in shaking flask, the DMEM culture mediums containing 10% NBCS is added, in 37 DEG C, 5%CO2Trained in incubator
Support, rotating speed is set to carry out suspension culture under the conditions of 140rpm/min, until cell density reaches 4 × 106Individual/mL.Other steps
It is rapid same as Example 3.
The full suspension cell culture of comparative example 2 produces PRV antigen
The step of full suspension cell culture of comparative example 2 produces PRV antigen is substantially the same manner as Example 3, difference
It is, the low blood serum medium of the step (2) and step (3) serum free medium are free of growth promoter.
The full suspension cell culture of comparative example 3 produces PRV antigen
The step of full suspension cell culture of comparative example 3 produces PRV antigen is substantially the same manner as Example 3, difference
It is, the growth promoter sulf onyl tamarind quality in the low blood serum medium of the step (2) and step (3) serum free medium
Oligosaccharide.
The full suspension cell culture of comparative example 4 produces PRV antigen
The step of full suspension cell culture of comparative example 4 produces PRV antigen is substantially the same manner as Example 3, difference
It is, the growth promoter only active mineral yeast in the low blood serum medium of the step (2) and step (3) serum free medium
Polypeptide.
The full suspension cell culture of comparative example 5 produces PRV antigen
The step of full suspension cell culture of comparative example 5 produces PRV antigen is substantially the same manner as Example 3, difference
Be, the growth promoter in the low blood serum medium of the step (2) and step (3) serum free medium by keratan sulfate and
Active mineral yeast polypeptides are with 1:0.15 mass ratio composition.
The full suspension cell culture of comparative example 6 produces PRV antigen
The step of full suspension cell culture of comparative example 6 produces PRV antigen is substantially the same manner as Example 3, difference
It is, the step (2) and low blood serum medium and serum free medium in step (3) are without polyglutamic acid.
The full suspension cell culture of comparative example 7 produces PRV antigen
The step of full suspension cell culture of comparative example 7 produces PRV antigen is substantially the same manner as Example 3, difference
It is, the step (2) and low blood serum medium and serum free medium in step (3) are commercially available prod, are purchased from moral
The ProBioGen companies of state.
Test example one, virus titer detection
According to《People's Republic of China's regulations》To embodiment 3-4 and the full suspension cells of comparative example 1-7
Culture production PRV titre is detected, as a result see the table below 3.
The virus titer testing result of table 3
Group |
Viral level (TCID50/mL) |
Embodiment 3 |
108.9 |
Embodiment 4 |
108.7 |
Comparative example 1 |
105.5 |
Comparative example 2 |
105.7 |
Comparative example 3 |
106.4 |
Comparative example 4 |
106.9 |
Comparative example 5 |
107.0 |
Comparative example 6 |
106.2 |
Comparative example 7 |
105.9 |
As seen from the above table, the full suspension cell culture production PRV titres of 3-4 of the embodiment of the present invention are higher, equal >
108.5TCID50/ mL, hence it is evident that better than the full suspension cell culture production PRV titres of comparative example 1-7.Can by comparative example 1
Know, after cultivating BHK21 cells using the DMEM culture mediums containing 10% NBCS, be continuing with containing 10% new born bovine
The DMEM culture mediums of serum carry out culture flask suspension culture to BHK21 cells, not can effectively improve the growth of BHK21 cells
State, is transferred to low serum free culture system environment by the BHK21 of flask suspension culture and carries out reactor suspension culture, the growth speed of cell
Degree is slow, and cytoactive declines, and low blood serum medium is unable to the growth of support suspension BHK21 cells continually and steadily.And add and contain 2
The nutrient solution of~4% NBCS carries out transition culture, can significantly improve growth shape of the BHK21 cells in low blood serum medium
State, so as to further improve culture virus titer.From comparative example 2-5 and 6, in low blood serum medium and serum free medium
It is middle to add a certain amount of growth promoter, the polyglutamic being made up of keratan sulfate oligosaccharide and active mineral yeast polypeptides respectively
Acid, can be effectively improved the state of BHK21 cell suspension growths, improve cell viability, reduce the appearance of dead cell, promote cell life
It is long, so as to further improve culture virus titer.
Test example two, antigen vaccine quality control
According to《People's Republic of China's regulations》3-4 of the embodiment of the present invention and comparative example 1-7 is made
PRV antigen vaccine carry out steriling test, mycoplasma examine, exogenous virus detection, as a result do not detect.
Test example three, safety evaluatio
PRV antigen finished product vaccine made from 3-4 of the embodiment of the present invention and comparative example 1-7 is pacified respectively
Full property evaluation, be specially:Choose 135 first 14~21 age in days piglets, be divided into 9 groups, every group 15, inject respectively embodiment 3-4 and
PRV antigen finished product vaccine made from comparative example 1-7, injection dosage is 4mL/ heads, and observation piglet is locally and systemically
Adverse reaction, as a result see the table below 4.
The safety evaluatio result of table 4
Result above shows, uses PRV antigen finished product made from 3-4 of the embodiment of the present invention and comparative example 1-6
Vaccine immunity piglet, the state of mind, feed intake and the body temperature of all immune piglets do not occur exception, also nonsystemic reaction, and
The vaccine produced using commercially available culture medium has 3 local red, swollen, scleroma phenomenon occur, has 2 of short duration temperature rise occur
(comparative example 7).The security that prompting purifying antigen of the present invention prepares vaccine is good.
Test example four, antibody induction experiment
PRV antigen finished product vaccine immunity made from the embodiment of the present invention 3 and comparative example 1-7 is respectively adopted small
Antibody level in mouse 21d, detection mice serum, and be compared with the antibody level in immune preceding mice serum, observation disease
The level of antibody is produced in malicious antigen induction body, 5 are as a result see the table below.
Mice serum antibody test result (ELISA OD after 21d are immunized in table 5450Value)
Note:Criterion, ELISA OD450> 0.6, then be the positive, ELISA OD450< 0.4, then be feminine gender.
As a result show, PRV antigen vaccine is remarkably improved in Mice Body made from the embodiment of the present invention 3
Antibody level, promotes to produce corresponding antibody in Mice Body, effect is better than comparative example 1-7, illustrates the antigen epidemic disease that the present invention is provided
Seedling can cause preferable immune response.
Test example five, attack poison protection evaluate
PRV antigen finished product vaccine immunity made from the embodiment of the present invention 3 and comparative example 1-7 is respectively adopted small
Mouse 21d, it is 0.45mL/ that every mouse, which attacks toxic agent amount, and mouse spleen, isolated viral are gathered after 21d days, glimmering using being immunized indirectly
Light (IFA) is detected that observation virus attacks the situation and protective rate of poison to mouse spleen, as a result see the table below 6.
The mice spleen of table 6 poison protecting effect
Group |
Total mice (only) |
The IFA positives (only) |
IFA feminine genders (only) |
Positive rate (%) |
Protective rate (%) |
Embodiment 3 |
15 |
0 |
15 |
0 |
100 |
Comparative example 1 |
15 |
8 |
7 |
53.3 |
46.7 |
Comparative example 2 |
15 |
7 |
8 |
46.7 |
53.3 |
Comparative example 3 |
15 |
6 |
9 |
40 |
60 |
Comparative example 4 |
15 |
5 |
10 |
33.3 |
66.7 |
Comparative example 5 |
15 |
4 |
11 |
26.7 |
73.3 |
Comparative example 6 |
15 |
6 |
9 |
40 |
60 |
Comparative example 7 |
15 |
7 |
8 |
46.7 |
53.3 |
The result that protest test is carried out to mouse shows PRV antigen epidemic disease made from the embodiment of the present invention 3
The immune protective rate of seedling reaches 100%, and vaccine potency evaluation criterion is fully achieved.
It the above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
The limitation of the present invention, protection scope of the present invention should be defined by claim limited range.For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, some improvements and modifications can also be made, these change
Enter and retouch and also should be regarded as protection scope of the present invention.